JP2003500056A - Regulation of protein conformation using molecular chaperones - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for regulating the conformation of a protein using a molecular chaperone. The chaperones of the present invention are applicable to a variety of protein conformational problems, including misfolding and aggregation. The molecular chaperone system of the present invention, including the DnaK system and ClpB, provides a particularly effective tool for regulating protein conformation. A further subject of the present invention is a novel cochaperone ClpS.

Description

Detailed Description of the Invention

The present invention relates to the use of molecular chaperones for the regulation of protein conformation,
The present invention relates to a novel chaperone mechanism and cochaperone. All patents, published patent applications, and other references cited herein are hereby incorporated by reference in their entireties.

[0002] The functional activity of proteins requires a balance between structural stability and flexibility, achieved by the low conformational stability of folded proteins. Absoluteness to this equilibrium is the correct and incorrect folding and the frequent separation of natural and unnatural structures by only small energy barriers (Jaenicke and Seckler, 1999), with slight changes in amino acid sequence or folding environment. Can dramatically affect protein folding and structural integrity. In fact, protein misfolding is the result of major damage in a variety of stress situations, including heat and oxidative stress (Bukau, 1999).
Misfolded proteins are generally exposed to hydrophobic surfaces and are therefore prone to intercellular aggregation, a process that has long been thought to be irreversible in vivo. Protein aggregation is biomedically important and has been implicated as the cause of several ring pathophysiological conditions including amyloidosis and prion disease (Horwich and Weissman, 1997, Lindquist, 1997,
Prusiner, 1997, Thomas et al., 1995, Wetzel, 1996). Moreover, aggregation of inclusion bodies with their attendants is of great importance in biotechnology as it is a frequent fate of recombinant proteins overproduced in host cells (Lilie et al., 1998, Mitraki and King, 1989).

One solution to protein misfolding and aggregation is to work with a protease that prevents the aggregation of misfolded proteins and assists in protein refolding or promotes degradation of the aggregation. Included are molecular chaperones that can be (Bukau, 1999, Hartl, 1996, Morimoto et al., 1994). H
Many chaperones, including sp90 (HtpG) and small HSPs (IbpA and IbpB), form functional networks and act as holders to prevent aggregation of misfolded proteins (E. coli homologs shown in brackets).
. Other chaperones (especially each cochaperone (for GroEL see Gro
In the case of ES and DnaK, Hsp60 (GroE containing DnaJ and GrpE)
L) and Hsp70 (DnaK) heat shock proteins) act as folders that not only prevent aggregation but also aid in refolding (Buchberger et al.,
1996, Ehrnsperger et al., 1998, Freeman et al., 1996, Johnson and Craig, 1997.
, Langer et al., 1992, Veinger et al., 1998). Other chaperones, especially eubacterial inducers, associate with ribosomes during biosynthesis to interact with early polypeptides. The role of inducers in protein folding in vivo has not been described in the literature.

A unique feature of the activity of chaperones is their ability to iteratively interact with protein folding intermediates to dissociate the substrate from the chaperone and either fold to its native state or reassociate with the same or another type of chaperone. is there. These substrate interaction cycles control many chaperones by ATP binding and hydrolysis.
Cells have been shown to have a limited ability to activate chaperones (Craig and Gross, 1991, Tatsuta et al., 1998, Tomoyasu et al., 1998), which assists the chaperone in the process of protein folding. It is suggested that it can be rate limiting during overproduction of and under pathophysiological stress of cells.

Many unknown aspects of chaperone activity have the potential to resolve pre-existing protein aggregations and mediate refolding. The E. coli DnaK chaperone has the ability to associate with its cochaperone and slowly dissociate E. coli heat-denatured RNA polymerase (Skowyra et al., 1990; Ziemienowicz et al., 1993). However, in all other reported cases, the Hsp70 family members show no or only little disaggregation activity, so this disaggregation activity is a variant of Dnak and Hsp7.
It appears to be another member of the 0 family (eg Schroder et al., 1993).
In contrast, members of the Hsp100 family of S. cerevisiae (Hsp104
The Hsp70 chaperone system is capable of refolding these enzymes (Glover and Lindquist, 1998, Parsell et al., 19 since heat shock proteins) have been shown to cleave heat-denatured firefly luciferase and β-galactosidase.
94).

Published evidence shows the high potential of molecular chaperones to assist the highly diverse protein folding process of cell lines. However, for published knowledge of the specific role of individual bacterial chaperones in protein aggregation protection and protein disaggregation and / or refolding in vivo, and on the general activity of bacterial chaperones in protein disaggregation and refolding. Information is limited.

The present invention relates to the use of cellular proteins (collectively referred to as molecular chaperones) that mediate the disaggregation and refolding of aggregated proteins and the refolding of misfolded proteins. The present invention further provides two E. coli chaperone systems (C) for mediating efficient disaggregation and refolding of a wide range of aggregation proteins.
lpB (Hsp100 protein) and Dnak system (including DnaK, Hsp
70 proteins))). This combination of chaperones is useful for preventing and reversing the formation of recombinant protein aggregates and inclusions during overproduction in E. coli and other host cells and for increasing the yield of soluble and active recombinant proteins . These chaperones can be used in vivo (especially but not exclusively E. coli) and in vitro. The present invention also provides
For example, methods of treating diseases caused by protein misfolding and aggregation using the molecular chaperone systems disclosed herein are provided.

Accordingly, in one aspect, the invention comprises a first protein comprising a target protein and at least one Hsp100 protein or homologue thereof for modulating the conformation of the target protein and at least a Hsp70 protein or homologue thereof. It is a method of regulating the conformation of a target protein by contacting it with a second protein.

The present invention provides a recombinant expression vector system. In one aspect, one recombinant expression vector encodes at least one Hsp100 protein or homologue thereof and at least one Hsp70 protein or homologue thereof. The expression vector can further encode one or more chaperones for the Hsp100 and Hsp70 proteins and one or both of the proteins of interest. In another aspect, one vector is used to express each protein component of the molecular chaperone system.

In another aspect, the invention provides a host cell transformed with a vector or vector system. In a preferred embodiment, host cells are engineered to recombinantly express the protein of interest and the molecular chaperone system described above. In the practice of the invention, inhibition of aggregation and misfolding of the protein of interest, for example by use of host cells so engineered, results in the protein of interest compared to cells in which the molecular chaperone system is not recombinantly expressed. It is possible to increase the yield of the active conformation of.

Another aspect of the present invention is to provide the aggregated protein with at least one Hsp100 protein or homologue thereof for deaggregating the aggregated protein and Hsp70.
An in vitro method of disaggregating aggregated proteins by contacting with a molecular chaperone system containing the protein or its homologue. In another aspect, the invention provides at least one Hsp10 for increasing the solubility of a recombinantly expressed protein.
A method of increasing the solubility of a recombinantly expressed protein by co-expressing a molecular chaperone system comprising a 0 protein or a homologue thereof and at least one Hsp70 protein or a homologue thereof is provided.

[0012] In another aspect, the invention provides a therapeutically effective amount of at least one Hsp100 protein or homologue thereof (preferably prokaryotic homologue) and Hsp70 protein or homologue thereof for preventing protein misfolding or aggregation. A method for preventing cell death due to protein misfolding or aggregation, comprising the step of administering a molecular chaperone system having (preferably a prokaryotic homolog).

The present invention also relates to animal diseases due to protein misfolding and aggregation, which treat the disease by administration of a therapeutically effective amount of a molecular chaperone system having at least one Hsp100 protein or homologue thereof and Hsp70 protein or homologue thereof. To provide a cure for The present invention also provides gene therapy for treating diseases using polynucleic acid molecules encoding the molecular chaperone system disclosed herein.

A further aspect of the present invention is an effective amount of a molecular chaperone system comprising at least one Hsp100 protein or homologue thereof (preferably prokaryotic homologue) and Hsp70 protein or homologue thereof (preferably prokaryotic homologue) as well as pharmaceutically. A pharmaceutical composition comprising an acceptable carrier.

Another aspect of the invention is to increase the efficiency of folding in vitro by adding an inducer to the in vitro protein synthesis system with or without at least one Hsp70 protein or homologue. Is a method of increasing the folding efficiency of the.

In another aspect, the invention is an isolated polynucleotide encoding a ClpS protein and derivatives thereof that is capable of modulating Hsp100 protein or homologues thereof in disaggregation. In one aspect, the invention provides polypeptides such as Clp and its derivatives that are capable of modulating Hsp100 homologs in disaggregation. A further aspect includes methods of modulating Hsp100 protein or homologues thereof in disaggregation.

The present invention also provides a molecular chaperone system for regulating a protein conformation comprising at least one Hsp100 protein or a homologous first protein thereof and a Hsp70 protein or a homologous second protein thereof.

DETAILED DESCRIPTION OF THE INVENTION Definitions Before describing the invention further, for convenience, certain terms used in the specification, examples, and appended claims are collected and described.

[0019]   "DNA" refers to single-stranded or double-stranded deoxyribonucleic acid.

“Complementary DNA” (cDNA) is a DNA having a nucleic acid sequence obtained by reverse transcription of messenger ribonucleic acid (mRNA).

“Recombinant gene expression” (also called recombination, gene recombination, or recombinant expression)
Refers to a method of transforming a host cell with a nucleic acid molecule that encodes the polypeptide or protein of interest so that the host cell can encode the polypeptide of interest. A plasmid or vector can be used to transfer the nucleic acid molecule into a host cell. The plasmid or vector may contain, in addition to the gene or nucleic acid sequence of interest, a gene expressing a selectable marker or phenotype and a gene capable of regulating (inducing or inhibiting) gene expression of interest under certain conditions. Yes, but not necessarily.

“Protein conformation” refers to the structure of a protein, including its folding and function. Normal proteins are generally active (ie, capable of performing normal function). An inactive protein is unable to perform its normal function or at its normal rate.
Protein inactivity can be the result of a number of factors, including misfolding.
Misfolded proteins can aggregate.

“Protein aggregation” refers to fibers, amorphous aggregates, inclusion bodies, dimers, trimers, or more than one in which at least one protein is misfolded or inactivated. It is another protein form of the protein.

“Soluble protein” is a protein that does not aggregate but can be misfolded.

A “molecular chaperone” is a protein that regulates, regulates, or assists in protein conformation. Molecular chaperones are proteins that can correctly fold, refold, prevent aggregation, or disaggregate proteins. An example of a molecular chaperone is DnaK, although many different types of chaperones exist within species as well as interspecies homologs.

A “cochaperone” is a protein that improves the ability of cochaperone to exert its function in protein conformation. Examples of cochaperones include ClpS and DnaJ and Grp that assist DnaK function.
E includes, but is not limited to. The latter are both preferred for the present invention.

“Molecular chaperone system” includes either a molecular chaperone and a cochaperone or a second chaperone. The Hsp70 and Hsp100 proteins are Lind
See US Pat. No. 5,827,685 to Quist for further details. As used herein, Hsp70 and Hsp100 “homologs” are generally present in various species and can be found in, for example, E. coli. Suitable prokaryotic homologs are ClpA, ClpB, and ClpxX. For example, ClpB is an E. coli Hsp100 protein homolog, which is particularly preferred for the present invention and more particularly corresponds to Hsp104. DnaK is the Escherichia coli Hsp70 protein. DnaJ and GrpE are E. coli DnaK cochaperones, homologues of which are present in monoprokaryotes and also in eukaryotes.

When referring to the proteins and nucleic acid molecules of the invention, the term “family” refers to
It is intended to mean two or more protein or nucleic acid molecules having a common structural domain and sufficient amino acid or nucleotide sequence homology as defined herein. Such family members are naturally occurring and can be from either the same or different species. For example, the family may include a first protein of human origin as well as another protein of human origin or may include homologues of non-human origin. Family members can also have common functional characteristics.

“DnaK system” refers to DnaK and the cochaperones DnaJ and GrpE.

The “GroEL-GroES system” is the Gso60 protein (GroEL) and its cochaperone (GroES).

“Protein of interest,” “target protein,” and “substrate” are used interchangeably and share the same meaning in that they refer to any protein or polypeptide. The "protein of interest" is often used when referring to in vivo, in vitro, isolated, or purified recombinantly expressed protein, but refers to any protein or polypeptide unless otherwise specified. The term "target protein" is often used to refer to any protein that should act during recombinant expression or synthesis,
Unless otherwise specified, it refers to any protein or polypeptide. A "substrate" is often used when referring to any protein or polypeptide that acts, but refers to any protein or polypeptide unless otherwise specified.

A “vector system” is a recombinant expression vector system. In one example, the recombinant expression vector can encode one or more proteins of interest. In another example, the vector can express one, two, three, etc. proteins. Host cells can be transformed with one or more vector systems. Preferred vector systems include Bujard and coworkers (Lutz and Bujard, 1997.
Cassette-like plasmid system, which was used to position the recombinant vector system.

As used herein, “polypeptide derivative / protein derivative” differs from a sequence described in the amino acid sequence, or a sequence in a sequence-free method, or both, and refers to a polypeptide or protein. Refers to a polypeptide / protein sequence with conserved activity. Derivatives of amino acid sequences are produced when one or more amino acids are replaced with different natural amino acids, amino acid derivatives, or unnatural amino acids. A particularly preferred embodiment is a naturally occurring polypeptide or protein whose sequence differs from the wild-type sequence by one or more conservative amino acid substitutions and which has minimal effect on the secondary structure and hydrophobicity of a typical protein or peptide. Or a biologically active fragment. Derivatives may also have sequences that differ by one or more non-conservative amino acid substitutions, deletions, or insertions that do not destroy the biological activity of the polypeptide or protein. Conservative substitutions (substituents) typically include the replacement of one amino acid with another amino acid having similar characteristics, such as substitutions within the following groups: valine, glycine; glycine, alanine; valine. , Isoleucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Non-polar (hydrophobic) amino acids include alanine, leucine,
It includes isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine.
Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

Other conservative substitutions can be listed in Table 1, but still other substitutions are Dayhoffi
n, The Atlas of Protein Sequence and Structure (1988).

[0035]

[Table 1]

Other derivatives of the invention are those with modifications that increase peptide stability. Such derivatives may include, for example, one or more non-peptide bonds (instead of peptide bonds) in the peptide sequence. Residues other than naturally occurring L-type amino acids (such as D-type amino acids) or non-natural or synthetic amino acids (β or α
Derivatives, including amino acids and cyclic derivatives, etc.) are also included. Incorporation of D-amino acids in the polypeptide in place of L-amino acids can increase protease resistance. See U.S. Pat. No. 5,219,990.

Polypeptides and proteins of the present invention may be modified by various alterations such as conservative or non-conservative insertions, deletions, and B substitutions, where the alterations may result in certain benefits in their use. You can also do it.

In another aspect, the desired derivatives can also be obtained by low-conservative amino acid substitution derivatives due to changes in charge, conformation, and other biological properties. Such substitutions include, for example, replacing a hydrophilic residue with a hydrophobic residue, cysteine or proline with another residue, or a residue with a small side chain to a residue with a large side chain. Or the substitution of a residue with a net positive charge for a residue with a net negative charge. If the outcome of a given substitution cannot be reliably predicted, the derivative can be rapidly assayed by the methods disclosed herein to identify the presence or absence of the desired characteristic.

Derivatives within the scope of the present invention include proteins and peptides having an amino acid sequence that has at least 80% homology to the polypeptides and proteins described herein. More preferably, the sequence homology is at least 90%,
Even more preferably at least 95%.

Just as it is possible to substitute the substituents of the skeleton, it is possible to substitute the functional groups which decorate the skeleton with groups which are characterized by similar characteristics. Such substitutions are initially conservative. That is, the substituent is of approximately the same size as the original group,
It has shape, hydrophobicity, and electric charge. Non-sequence modifications include, for example, in vivo or in vitro chemical derivatives of portions of naturally occurring polypeptides or proteins,
And acetylation, methylation, phosphorylation, carboxylation, or glycosylation.

In a further aspect, the protein is modified by a chemical modification that preserves activity. For example, the protein can be amidated, sulfated, single or multiple halogenated, alkylated, carboxylated, or phosphorylated. 1 of protein
One or more may be acylated with an acetyl group, a farnesyl group, or a fatty acid capable of being saturated, monounsaturated or polyunsaturated. One or more of the fatty acids can be fluorinated. The invention also includes methionine analogs of proteins such as methionine sulfone and methionine sulfoxide analogs. The present invention also includes protein salts (ammonium salts including alkyl or aryl ammonium salts, sulfates, hydrogen sulfates, phosphates, hydrogen phosphates, dihydrogen phosphates, thiosulfates, carbonates, hydrogen carbonates). Salts, benzoates, sulfonates, thiosulfonates, mesylates, ethyl sulfonates, benzene sulfonates and the like).

Derivatives of polypeptides and proteins can also include peptidomimetics.
Such compounds are well known to those of skill in the art and include, for example, compounds produced by the substitution of certain R groups or amino acids in proteins that include non-physiological, non-natural substitutions. Such substitutions may increase the stability of such compounds over naturally occurring compounds.

Recombinant expression vectors containing a nucleic acid sequence encoding a recombinant polypeptide or protein can be prepared using well known methods. Expression vectors include a DNA sequence operably linked to appropriate transcriptional or translational regulatory nucleotide sequences from mammalian, bacterial, viral, or insect genes. Examples of regulatory sequences include transcription promoters, operators, or enhancers, mRNA ribosome binding sites, and appropriate sequences that regulate transcription and translation initiation and termination.
A nucleotide sequence is "operably linked" when the regulatory sequence relates to a DNA sequence that encodes the polypeptide or protein of interest. For example, a promoter nucleotide sequence is operably linked to a DNA sequence encoding a protein or polypeptide of interest if the nucleotide sequence of the promoter controls transcription of the DNA sequence encoding the protein of interest. The origin of replication and the ability to replicate in the desired host cell, usually conferred by the selection gene by which the transformants are identified, can be additionally incorporated into the expression vector.

In addition, sequences encoding appropriate signal peptides that do not naturally associate with the polypeptide or protein can be incorporated into expression vectors. For example,
The DNA sequence of the signal peptide (secretory leader) can be fused in frame to the DNA sequence of interest so that the protein of interest is first translated as a fusion protein containing the signal peptide. A signal peptide that is functional in the intended host cell enhances extracellular secretion of the polypeptide or protein of interest. Upon secretion of the cell-derived polypeptide or protein, the signal peptide can be cleaved from the polypeptide or protein.

Suitable expression host cells include prokaryotic, yeast, or higher eukaryotic cells. Suitable cloning and expression vectors for bacterial, fungal, yeast, and mammalian cell hosts are described, for example, in Pouwels et al., "Cloning Vectors: A Laboratory Manual," Elsevier, New Yo.
rk, 1985. Cell-free translation systems using RNA from the DNA constructs disclosed herein can also be used to produce polypeptides or proteins.

Prokaryotes (also prokaryotes) are Gram-negative or Gram-positive organisms (eg,
E. coli or Bacilli). Suitable prokaryotic host cells for transformation include
For example, E. coli, Bacillus subtilis, Salmonella typhimurium, and Pseu.
Includes domonas, Streptomyces, and various other species within the Staphylococcus genus. In prokaryotic host cells (such as E. coli), the polypeptide or protein may include an N-terminal methionine residue that facilitates recombinant polypeptide expression in a prokaryotic host cell. The N-terminal Met can be cleaved from the expressed polypeptide or protein.

Expression vectors for use in prokaryotic host cells generally include one or more phenotypic selectable marker genes. The phenotypic selection marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or supplies autoauxotrophy. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids (cloning vector pBR322 (ATCC3
7017) etc.). Since pBR322 contains ampicillin and tetracycline resistance genes, a simple means for identifying transformed cells can be obtained. To construct an expression vector using pB322, a promoter and a DNA sequence encoding a polypeptide or protein is inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and pGEMI (Promega Biotec, Madison, Wis., USA)
) Is included.

Commonly used promoter sequences for recombinant prokaryotic expression vectors include 1
3-lactamase (Penicillinase), lactose promoter system (Chang et al.,
Nature, 275: 615, 1978 and Goeddel et al., Nature, 281, 544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res., 8 :).
4057, 1980, and European Patent Application No. 36776), and the tac promoter (Ma
niatis, "Molecular Cloning: A Laborat
ory Manual) ”, Cold Spring Harbor Laboratory, p412, 1982).
Particularly useful prokaryotic host cell expression systems include the phage LPL promoter and c
The I857ts thermolabile repressor sequence is used. The plasmid vector available from the American Type Culture Collection in which a derivative of the LPL promoter is incorporated includes plasmid pHUB2 (Escherichia coli JMB9 strain (ATCC3709).
2)) and pPLc28 (present in E. coli RR1 strain (ATCC 53082)). See, for example, Example 6 below, which describes the exemplification of the invention discussed for cassette-like plasmid systems.

Alternatively, the polypeptides and proteins of the invention can be expressed in yeast host cells, preferably from the genus Saccharomyces (eg S. cerevisiae). Other yeast genera (Pichia ), Klactis, or Klayreromyces .. Yeast vectors are often derived from the 2μ yeast plasmid, which is an origin of replication.
It contains an autonomously replicating sequence (ARS), a promoter region, a polyadenylation sequence, a transcription termination sequence, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, for example, metallothionein, 3-phosphoglycerate kinase (Hitzem
an et al., J. Biol. Chem., 255, 2073, 1980), or other glycolytic enzymes (Hess et al., J. Ad.
v. Enzyme Reg., 7, 149, 1968 and Holland et al., Biochem., 17, 4900, 1978.
) (Enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phospho Glucose isomerase, and glucokinase, etc.) promoters. Other suitable vectors and promoters for yeast expression are Hitzeman, European Patent Application No. 73657, or Fleer.
Et al., Gene, 107, 285-195, 1991, and van den Berg et al., Bio / Technology,
8, 135-139, 1990. Another choice is Russell
J. Biol. Chem., 258, 2674, 1982 and Beier et al., Nature, 300, 724, 19.
82. The glucose-repressed ADH2 promoter described in 82. Both yeast and E. coli replicable shuttle vectors can be constructed by insertion of the pBR322 derived DNA sequences (Ampr gene and origin of replication) into the yeast vector described above for selection and replication in E. coli.

The yeast alpha factor leader sequence can be used to direct secretion of a polypeptide or protein. The alpha factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. For example, Kurjan et al., Cell, 30, 933, 1982,
See Bitter et al., Proc. Natl. Acad. Sci. USA, 81, 5330, 1984, U.S. Pat. No. 4,546,082, and European Patent 324,274. Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art. The leader sequence can be modified near its 3'end to include one or more restriction sites. This facilitates fusion of the leader sequence to the structural gene.

Yeast transformation protocols are known to those of skill in the art. One such protocol is described in Hinnen et al., Proc. Natl. Acad. Sci. USA, 75, 1929, 1978. In the Hinnen et al. Protocol, Trp + transformants are selected in a selection medium consisting of 0.67% yeast nitrogen source, 0.5% casamino acid, 2% glucose, 10 μg / ml adenine, and 20 μg / ml uracil.

Yeast host cells transformed with a vector containing the ADH2 promoter sequence can be grown for inducible expression in "enriched" media. An example of an enriched medium is one containing 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 μg / ml adenine and 80 μg / ml uracil. When glucose is depleted from the medium, the ADH2 promoter is derepressed.

Mammalian or insect host cells can also be used to express the polypeptides or proteins of the invention. Baculoviruses for the production of heterologous proteins in insect cells have been reviewed by Luckow and Summers, Bio / Technology, 6, 47, 1988. Established cell lines of mammalian origin can also be used. Examples of suitable mammalian host cell lines include monkey kidney cell line COS-7 (ATCC CRL1).
651) (Gluzman et al., Cell, 23, 175, 1981), L cells, C127 cells, 3T.
3 cells (ATCC CCL 163), Chinese hamster ovary (CHO)
Cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and
The CV-1 / EBNA-1 cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) described in McMahan et al., EMBO J., 10, 2821, 1991 is included.

Transcriptional and translational regulatory sequences for mammalian host cell expression vectors can be excised from viral genomes. Commonly used promoter and enhancer sequences are polyomavirus, adenovirus 2, simian virus 4
0 (SV40), and human cytomegalovirus. DNA sequences from the SV40 viral genome (eg, SV40 origin of replication, early and late promoters, enhancers, splices and polyadenylation sites) can be used to obtain genetic elements for expression of structural gene sequences in mammalian host cells. . The viral early and late promoters are particularly useful because they can be easily obtained from the viral genome as fragments that may also contain the viral origin of replication (Fiers et al., Nature, 273, 113, 1978). Small or large SV40 fragments can also be used if they contain an approximately 250 bp sequence extending from the HindIII site towards the BgI site present in SV40.

Exemplary expression vectors for mammalian host cells disclosed in Okayama and Berg, Mol. Cell. Biol., 3, 280, 1983 can be constructed. A useful system for stable high level expression of mammalian cDNA in C127 mouse mammary epithelial cells can be fully constructed as described by Cosman et al., Mol. Immunol., 23., 935, 1986. Useful high expression vector PMLS described in Cosman et al., Nature, 312, 768, 1984.
V N1 / N4 has been deposited as ATCC 39890. Further useful mammalian expression vectors are described in European Patent Application 0367566. The vector may be from a retrovirus. Instead of the native signal sequence, a heterologous signal sequence (eg IL-7 signal sequence described in US Pat. No. 4,965,195, Cosman
Et al., Nature, 312, 768, 1984, the signal sequence of the IL-2 receptor, the IL-4 signal peptide described in EP 0367566, U.S. Pat. No. 4,968,607.
IL-1 receptor signal peptide as described in US Pat.
II type IL-1 receptor signal peptide described in No. 846) can be added.

Purification and Isolation Methods The isolated and purified polypeptides or proteins of the invention can be produced by the recombinant expression systems described above or purified from naturally occurring cells. A polypeptide or protein can be substantially purified as displayed by a single band of protein in SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis. One of the methods for producing a polypeptide or protein of the present invention is a host cell transformed with an expression vector containing a DNA sequence encoding the polypeptide or protein under conditions sufficient to promote the expression of the polypeptide or protein. And culturing. The polypeptide or protein is then recovered from the culture medium or cell extract, depending on the expression form used. As is known to those of skill in the art, purification procedures for recombinant proteins will vary depending on such factors as the type of host cell used and whether the recombinant protein is secreted into the culture medium. For example, when using an expression system that secretes a recombinant protein, the overview medium is first added to a commercially available protein concentration filter (eg Amicon or Millipor).
e Pellicon ultrafiltration unit) can be used to concentrate. After concentration, the concentrate can be applied to a purification matrix such as gel filtration media.

Alternatively, an anion exchange resin can be used, such as a matrix or substrate with pendent diethylaminoethyl (DEAE) groups. The matrix is acrylamide, agarose, dextran, cellulose,
Or it may be another type commonly used for protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include various insoluble carriers containing sulfopropyl or carboxymethyl groups. A sulfopropyl group is preferred. Finally, one or more reverse phase high performance liquid chromatographies (RP-HPLC) using hydrophobic RP-HPLC means (eg silica gel with attached methyl or other aliphatic groups) are used to analyze the poly The peptide or protein can be further purified. Combinations of some or all of the above purification steps are well known and can be used to obtain isolated and purified recombinant proteins.

To affinity purify the expressed polypeptide or protein, affinity columns containing binding proteins specific for the polypeptide or protein of the invention are available. Polypeptides or proteins can be labeled by conventional techniques (eg, elution with high salt elution buffer followed by dialysis against the low salt buffer used or changing pH or other components depending on the affinity matrix utilized). Can be used to recover from the affinity column.

Recombinant proteins produced in bacterial cultures are usually first disrupted by host cells, centrifuged, extracted from cell pellets for insoluble polypeptides or supernatants for soluble polypeptides. Isolation by extraction followed by one or more centrifugation, salting out, ion exchange, affinity purification, or size exclusion chromatography steps. Finally, RP-HPLC can be performed as the final purification step. Bacterial cells can be disrupted by any conventional method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

To simplify purification, transformed yeast host cells can be used to express the polypeptide or protein as a secreted polypeptide. Secreted recombinant polypeptides derived from yeast host cell fermentates have been described by Urdal et al., J. Chromatog., 296, 171.
, 1984. Urdal et al. Describe two consecutive reverse phase HPLC steps for purification of recombinant human IL-12 by preparative HPLC column.

Nucleic Acid According to the present invention, a single strand capable of binding to a target mRNA sequence (double helix formation) or double stranded DNA helix (triple helix formation) encoding a polypeptide or protein of interest. Antisense or sense oligonucleotides containing nucleic acid sequences (either RNA or DNA) can be made. According to the invention, the antisense or sense oligonucleotide is a cDNA
It includes a polypeptide or protein fragment encoding the A region. Such fragments generally contain at least 14 nucleotides, preferably about 14-30 nucleotides. The ability to generate antisense and sense oligonucleotides based on the cDNA sequence of a given protein is eg
Stein and Cohen, Cancer Res., 48, 2659, 1988 and van der Krol et al., Bi.
oTechniques, 6, 958, 1988.

Attachment of an antisense or sense oligonucleotide to a target nucleic acid sequence results in one of several means, including enhanced double helix degradation, premature termination of transcription or translation, or other means. Translation (RNA) or transcription (DNA)
A complex that blocks is formed. Therefore, antisense oligonucleotides can be used to interfere with the expression of the polypeptides or proteins of the invention. The antisense or sense oligonucleotide further has a modified sugar phosphodiester backbone (or other sugar bond (bond described in WO91 / 06629)),
Such sugar linkages include oligonucleotides that are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo (ie, may be resistant to enzymatic degradation) but retain sequence specificity capable of binding the target nucleotide sequence. Other examples of sense or antisense oligonucleotides include covalently linked organic moieties (described in WO90 / 10448) and other moieties that increase the affinity of the oligonucleotide for target nucleic acid sequences (such as poly (L-lysine)). Oligonucleotides that are included. In addition, intercalating agents (elipticin) and alkylating agents or metal complexes can be attached to the sense or antisense oligonucleotides to modify the binding specificity of the antisense or sense oligonucleotides of the target nucleotide sequence. .

Cells containing antisense or sense oligonucleotides containing the target nucleic acid sequence by any gene transfer method (including CaPO ₄ -mediated DNA transfection, electroporation) or the use of gene transfer vectors such as Epstein-Barr virus. Can be introduced to. An antisense or sense oligonucleotide is added to a cell containing the target nucleic acid sequence by inserting the antisense or sense oligonucleotide into a suitable retroviral vector, and then contacting the cell in vivo or ex vivo with the retroviral vector containing the inserted sequence. It is preferable to transfer. Suitable retroviral vectors include, but are not limited to, murine retroviruses M-MuLV, N2 (M-MuLV-derived retrovirus), or double copy vectors called DCT5A, DCT5B, and DCT5C.

Sense or antisense oligonucleotides can also be introduced into cells containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule as described in WO91 / 04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, the conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind its corresponding molecule or receptor or to interfere with the entry of sense or antisense oligonucleotides or binding versions thereof into cells.

Alternatively, sense or antisense oligonucleotides can be introduced into cells containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex as described in WO90 / 10448. Sense or antisense oligonucleotide-
The lipid complex is preferably dissociated intracellularly by endogenous lipase.

The present invention is directed to stringent conditions, especially highly stringent conditions,
Includes polynucleotides that can hybridize to the polynucleotides described herein. Highly stringent conditions include, for example, 0.2 × at 65 ° C.
SSC is included and stringent conditions include, for example, 4xSSC or 50% formamide at 65 ° C and 4xSSC at 42 ° C. Preferably, such hybridizing nucleotides are at least 70% identical (more preferably at least 80% identical, even more preferably 85%, 90%, or 95% identical) to the hybridizing polypeptide of the invention. is there.

The invention also relates to naturally occurring isolated polynucleotides that also encode allelic variants of the disclosed polynucleotides or proteins (ie, proteins that are identical, homologous, or related proteins to those encoded by the polynucleotides of the invention. Present or non-naturally occurring alternatives).

Homology or Identity To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (eg, first and second for optimal alignment). Gaps can be inserted in one or both of the amino acid or nucleic acid sequences of
Heterologous sequences can be ignored for comparison). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably the length of the reference sequence. Is at least 70%, 80%, 90%, or 95%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (the amino acid or nucleic acid "identity" used herein). Is equivalent to an amino acid or nucleic acid "homology"). The percent identity between two sequences is 2
It is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that must be introduced for optimal alignment of the two sequences, the length of each gap.

Sequence comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between the two amino acid sequences
Blossom 62 matrix or PAM250 matrix, 16,
Gap weights of 14, 12, 10, 8, 6, or 4 and 1, 2, 3, 4
Need embedded in the GAP program in the GCG software package (available from http://www.gcg.com) using length waits of 5, or 6
Identification using the leman and Wunsch (J. Mol. Biol., 48, 444-453, 1970) algorithm. In a further preferred alternative embodiment, the% identity between the two nucleotide sequences is determined by NWSgapdna. Identify using the GAP program in the GCG software package (available from http://www.gcg.com) using weights. In another aspect, the percent identity between two amino acid or nucleotide sequences is determined by the PAM120 weight residue table, 1
A with a gap length penalty of 2 and a gap penalty of 4
E.Meyers and W.Miller, CABIOS, 4, 11-17, 1989 algorithms embedded in the LUGN program (available at http://vega.jgh.cnrs.fr/bin/align-guess.cgi) Use to identify.

The nucleic acid and protein sequences of the invention can be used as "search sequences" to further search public databases to identify, for example, other family members or related sequences. Such searches are described in Altschul et al., 1990, J.
Mol. Biol., 215, 403-1O NBLAST and XBLAST programs. To obtain a nucleotide sequence homologous to the MSP-18 nucleic acid molecule of the present invention, the NBLAST program (score = 100, string length = 1
BLAST nucleotide searches can be performed using 2). MSP of the present invention
BLAST protein searches can be performed using the XBLAST program (score = 50, string length = 3) to obtain amino acid sequences homologous to -18 protein molecules. To obtain a gap alignment for comparison, Altschul et al.
1997, Nucleic Acid Res., 25 (17), 3389-3402, GAP BLAST.
Can be used. When utilizing BLAST and GAP BLAST programs, the default parameters for each program (eg, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Furthermore, the "Clustal" method (Higgins and Sharp, Gene, 73, 237-
44, 1988) and the "Megalign" program (Clewley and Arnold, Methods Mo.
l.Biol., 70, 119-29, 1997) can be used to align sequences to identify similarities, identities, or homologies. See Example 10 of the Invention below.

Gene Therapy The nucleic acid molecule of the present invention can be inserted into a vector and used as a vector for gene therapy. Gene therapy vectors may be administered, for example, by intravenous injection, topical administration (see US Pat. No. 5,328,470), or stereotactic injection (eg, Chen et al.,
1994, Proc. Natl. Acad. Sci. USA 91, 3054-3057)). The pharmaceutical preparation of the gene therapy vector can include a sustained release matrix incorporating the gene therapy vector or gene delivery agent in an acceptable diluent. Alternatively, the complete gene delivery vector can be produced intact from recombinant cells (eg, a retroviral vector) and the pharmaceutical preparation can include one or more cells that provide the gene delivery system. The pharmaceutical composition can be included in a container, package, or dispenser with instructions for administration.

The present invention provides a molecular chaperone having a target protein and a first protein of at least one Hsp100 protein or homolog thereof and a second protein of Hsp70 protein or homolog thereof to control the conformation of the target protein. Disclosed is a method for controlling the conformation of a target protein by contacting the system. In a preferred embodiment, the first protein comprises a prokaryotic homologue of Hsp100 protein, more preferably the homologue is a Clp homologue. The Clp homolog is preferably ClpA, ClpX, more preferably Clp.
pB. In a more preferred embodiment, the second protein is a prokaryotic homologue of Hsp70 protein. More preferably, the prokaryotic homolog is DnaK. In a further preferred embodiment, the molecular chaperone system further comprises at least one cochaperone. Preferred cochaperone is DnaJ, Grp
E, or ClpS, more preferably DnaJ and GrpE. Particularly preferred cochaperones are DnaJ and GrpE.

In a further aspect, the target protein may be recombinantly expressed in a host cell and the target protein aggregated due to overexpression in the host cell. In a more preferred embodiment, the first (Hsp100 protein) and the second (Hsp70 protein) are recombinantly expressed in the host cell. The host cell is preferably E. coli, S.
． S. Cerevisiae, B. subtilis, plant cells, insect cells,
It is a yeast cell or a mammalian cell. More preferably, the host cell is E. coli.

According to the present invention, a recombinant expression vector system is obtained. In one aspect, one recombinant expression vector encodes at least one Hsp100 protein or homologue thereof and at least one Hsp70 protein or homologue thereof. The expression vector can further encode one or more chaperones of one or both Hsp100 and Hsp70 proteins, along with the protein of interest. In another aspect, one vector is used to express each protein component of the molecular chaperone system. In another aspect, one, two,
Hsp100 proteins, such as 3, also like Hs 100, 2, 3, etc.
The p70 protein and all combinations with chaperone and cochaperone proteins in between can be expressed. Host cells can be transformed with one or more vectors of the system. In a preferred embodiment, Bujard et al. (Lutz
And a cassette-like plasmid system developed by Bujard, 1997),
Deploy the recombinant vector system. More preferably, the vector system is further capable of recombinantly expressing the GroEL-GroES system or a homolog thereof, each protein or homolog thereof, an inducer (TriggerFactor) or a homolog thereof. For a discussion of the GroEL-GroES system, see Bukau and Horwich,
Can be found in Cell, 1998.

In the present invention, target proteins can be aggregated or misfolded. The target protein may aggregate due to stress conditions (heat stress, oxidative stress, etc.), overexpression, mutation, or disease. In a preferred embodiment, the target protein is cleaved in vitro.

The invention further provides a vector system encoding a molecular chaperone system comprising at least one Hsp100 protein or homologue thereof and at least one Hsp70 protein or homologue thereof. Vector systems can include inducible promoters so that expression can be regulated with or without stimulation.

The vector system can be used to transform cells. In a preferred embodiment, the cells are protease deficient. More preferably, the protease deficiency is rpo
Caused by a mutation in the H gene, more preferably the mutation is GroEL
-A chromosomal deletion in the rpoH gene in cells which additionally has an IS element in the promoter region of the GroES operon, which allows cell proliferation.

In one aspect of the invention, the host cell is capable of recombinantly expressing the protein of interest. The protein of interest is probably misfolded or aggregated in the absence of recombinant expression of the molecular chaperone system. In another aspect, the expressed molecular chaperone system inhibits aggregation or misfolding of the protein of interest. In a preferred embodiment, aggregation and misfolding of the protein of interest inhibits an increase in the yield of the active conformation of the protein of interest compared to cells that do not recombinantly express the molecular chaperone system. In another aspect, the molecular chaperone system disaggregates and refolds aggregated proteins. In a preferred embodiment, the protein of interest is malate dehydrogenase. According to the invention, the cell may be any prokaryotic or eukaryotic cell, but more preferably the cell is E. coli S. Cerevisiae,
B. Subtilis, plant cell, insect cell, yeast cell, or mammalian cell.

The present invention further comprises an aggregated protein for disaggregating the aggregated protein,
At least one Hsp100 protein or homologue thereof and Hsp70
Provided is an in vitro method of disaggregating aggregated proteins by contact with a molecular chaperone system having the protein or a homologue thereof. In a preferred embodiment, the method comprises the step of folding the disaggregated protein into an active form.

In another aspect, the invention provides at least one Hsp100 protein or homologue thereof and Hsp7 for increasing the stability of a recombinantly expressed protein.
A method for increasing the stability of a recombinantly expressed protein by co-expressing a molecular chaperone system having 0 protein or a homologue thereof is provided.

In yet another aspect, the invention provides a therapeutically effective amount of a molecular chaperone system having at least one Hsp100 protein or homologue thereof and Hsp70 protein or homologue thereof to prevent protein misfolding and aggregation. It is a method of preventing cell death due to protein misfolding and aggregation by administration.

The present invention also relates to the treatment of a disease by the administration of a therapeutically effective dose of a molecular chaperone system having at least one Hsp100 protein or homologue thereof and Hsp70 protein or homologue thereof in animals by misfolding and aggregation of proteins. Provide a cure for the disease. In a preferred embodiment, the animal is a mammal such as a human, rodent, cat, or dog. Diseases that can be treated by the present invention include Creutzfeldt-Jakob disease, Alzheimer's disease, Huntington's disease, ataxia type 1, cystic fibrosis, and cancer. It is preferable to deliver the therapeutically effective dosage together with a pharmaceutically acceptable carrier. More preferably, the pharmaceutically acceptable carrier is capable of targeting misfolded proteins. Furthermore, the present invention provides gene therapy methods and compositions useful for performing gene therapy with a polynucleic acid molecule encoding a molecular chaperone system as described above.

A further aspect of the invention is a pharmaceutical composition comprising an effective dosage of a molecular chaperone system having at least one Hsp100 protein or homologue thereof and Hsp70 protein or homologue thereof and a pharmaceutically acceptable carrier.

Another aspect of the invention is that in vitro by the addition of an inducer with or without at least one Hsp70 protein or homologue thereof to the in vitro protein synthesis system to increase the folding efficiency in vitro. This is a method for increasing the folding efficiency of proteins. In a preferred embodiment, in addition to the inducer, a molecular chaperone system containing at least one Hsp70 protein or homologue thereof is added to the in vitro synthesis system. Recombinant expression in a cell of an inducer with or without at least one Hsp70 protein or its homologue can increase the folding efficiency of the protein in the cell, thereby increasing the folding efficiency in the cell. it can. The cell can be a prokaryotic cell or a eukaryotic cell. In a preferred embodiment, in addition to the inducer, at least one Hsp
A molecular chaperone system containing 70 proteins or homologues thereof is expressed recombinantly.

The present invention provides a polynucleic acid molecule related to the polynucleotide sequence of SEQ ID NO: 1. In one aspect, the invention provides (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, (b) a polynucleotide antisense to the polynucleotide of (a), (c) (a) or (b). A polynucleotide that is 80% identical to the polynucleotide of (4), (d) a polynucleotide that encodes the protein of SEQ ID NO: 2, (e) a polynucleotide that encodes a polypeptide that is 80% identical to the protein of SEQ ID NO: 2, (f ) A nucleic acid of (a) to (e), which encodes a protein capable of controlling the disaggregation activity of Hsp100 protein or a homolog thereof, and 8
0% identical polynucleotide, and (g) under stringent conditions (a)
There is provided an isolated polynucleic acid molecule selected from the group consisting of polynucleotides that hybridize to any one of the polynucleotides specified in (f). In a preferred embodiment, the polynucleotide of the invention is at least 70% identical, more preferably at least 80% identical, even more preferably 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO: 1.

The invention further provides polypeptides and proteins related to SEQ ID NO: 2. In one aspect, the invention provides: (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; (b) a polypeptide that is 80% identical to the amino acid sequence of SEQ ID NO: 2; Amino acid sequence polypeptide variant, (d) Hsp10
SEQ ID NO: which is capable of controlling the disaggregation activity of protein 0 or its homologues:
A polypeptide variant of the amino acid sequence of 2 (e) ClpA may be mediated
of cochaperoning) SEQ ID NO: 2, polypeptide variant of the amino acid sequence, (f)
A polypeptide encoded by the polynucleotide of SEQ ID NO: 1; (g) a polypeptide encoded by a polynucleotide that is 80% identical to the sequence of SEQ ID NO: 1; and (h) SEQ ID NO: under stringent conditions. There is provided an isolated polypeptide selected from the group consisting of a polypeptide encoded by a polynucleotide that hybridizes to a polynucleotide having the sequence of 1. In one aspect, the variants and derivatives are insertion, deletion, or substitution variants or derivatives. In a preferred embodiment, the polypeptide of the invention is at least 70% identical, more preferably at least 80% identical, even more preferably 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO: 2.

Furthermore, a molecular chaperone system is provided for controlling the conformation of a protein having at least one Hsp100 protein or a homologous first protein thereof and at least one Hsp70 protein or a homologous second protein thereof. To be done.

A further aspect is the use of ClpS or a homologue or derivative thereof to regulate the activity of the Hsp100 protein. In a preferred embodiment, Hsp10
0 protein is ClpA, ClpB, or ClpX. In a more preferred aspect, the Hsp100 protein is ClpA.

A further embodiment is protein folding in E. coli as well as in vitro protein synthesis systems using mutationally altered content of cytosolic chaperones (eg in mutants lacking the regulatory gene rpoH). It is a method of increasing efficiency.

In this regard, the DnaK (Hsp70 protein) chaperone system (DnaK,
The inducer (TF) in combination with DnaJ, consisting of GrpE) is important for protein folding and identified the cytosol of these two chaperone systems.

Furthermore, ClpB (Hsp100 protein) in combination with the DnaK system is important for protein folding, and is refolded at 30 ° C. after aggregation. D
The naK system along with ClpB is more suitable for increasing the viability of E. coli under severe heat shock conditions.

Furthermore, the DnaK and GroEL systems do not act primarily in a continuous fashion in assisting the folding of newly synthesized proteins, but rather the folding of significantly different substrate populations.

Another result of the present invention is that Lon and ClpXP are the major cytoplasmic substrate proteases that can evacuate aggregated proteins that accumulate in chaperone gene variants.

Two strains were used in the experiments performed to substantiate the findings. First, dna replaced by an artificial operon whose expression can be regulated by the inducer IPTG
TF-deficient tig :: kan strain further carrying the K dnaJ operon (Deue
rling et al., 1999). Second, the rpoH strain lacking the heat shock transcription factor σ32, which is responsible for the expression of all major cytosolic chaperones and proteases except TF. This published strain (Kusukawa and Yura, 1988) is viable at 30 ° C and 37 ° C as it is capable of constitutive expression of GroEL at high levels due to mutation of the repressor. Findings using these strains and these newly constructed derivatives are shown below.

About 10% of all cytosolic proteins were deficient in DnaK at 37 ° C. tig:
: Found in insoluble pellet in kan cells (lacking TF). This indicates that the DnaK / TF requirement is temperature dependent, as the amount of aggregated protein is smaller (1.2%) at 30 ° C. This finding indicates that the TF and DnaK systems are more important at higher growth temperatures for efficient protein folding in E. coli. These also suggest that the folding efficiency of recombinant proteins synthesized in E. coli-based in vivo and in vitro systems can be improved by supplementing the TF and / or DnaK system when the synthesis is performed at higher temperatures.

Furthermore, DnaK-depleted 3 by two-dimensional gel electrophoresis and subsequent mass spectrometry.
Large-scale identification of aggregated proteins identified at 7 ° C in tig :: kan cells was performed. Over 92 proteins have been identified that are involved in a variety of basic cellular processes including transcription, translation, and metabolism. A common salient feature is the size dependence of proteins of this type that can cover the entire molecular range (ie, several Da to several hundred kDa).
Particularly high molecular weight proteins formed by multiple domains require the assistance of TF and / or DnaK for productive folding. Similar to the heterologous proteins, the identified E. coli proteins were found to increase TF and DnaK yields of native recombinant proteins under high level production conditions in biotechnology applications.
Overproduction of the system is recommended. Examples of suitable proteins called aggregation-prone proteins are glutamate synthase (molecular weight about 167 kDa), RNA polymerase (about 150-160 kDa), Thr-tRNA synthetase (about 75 kDa).
Is.

In assisting the folding of newly synthesized proteins in E. coli cells, Dn
There is a functional association between the aK system, the GroEL system (consisting of GroEL and GroES) and TF. This association was investigated by alterations in cellular levels of independent or combined chaperones and analysis of chaperone-substrate interactions by co-immunoprecipitation with chaperone-specific antibodies.

Previously published experiments have previously shown that the TF and DnaK systems jointly perform protein folding (Deuerling et al., 1999, Teter et al., 19).
99). In strains with the dnaK dnaJ operon controllable by IPTG (
It was found that i) a deletion of the DnaK gene or (ii) a deletion of DnaK and DnaJ was sufficient for increased protein aggregation compared to wild type cells. These findings indicate that there is already another contribution of the DnaK system to protein folding that cannot be compensated for by the presence of other cellular chaperones. However, consistent with earlier findings (Deuerling et al., 1999), stronger protein aggregation occurred in tig cells lacking DnaK and DnaJ.

To analyze the functional association between DnaK and GroEL, an rpoH mutant containing a suppressive mutation capable of expressing GroEL was used. In this strain, about 5% of the total soluble cell protein was found to aggregate at 30 ° C. IPTG-controlled overproduction of GroEL and GroES from multicopy plasmids did not significantly reduce the amount of aggregated protein. In contrast, IPTG-induced overproduction of the DnaK system was sufficient to efficiently suppress aggregation of almost all aggregation-prone proteins. Concomitant production of both the DnaK system and ClpB was even more efficient at inhibiting protein aggregation at 30 ° C as well as after heat shock at 42 ° C and 50 ° C.

In addition, an in vivo substrate population associated with GroEL and DnaK in rpoH cells containing different levels of the plasmid-encoded DnaK system was identified. Gro
This identification was performed by co-immunoprecipitation of radiolabeled cellular proteins using EL or DnaK specific antisera. The result was that the substrate populations of DnaK and GroEL were significantly different but were unable to eliminate the duplication of these populations. Therefore, earlier findings (Deuerling et al., 1999, Ewalt et al.,
In 1997) GroEL has an upper substrate size limit of approximately 65 kDa. In contrast, the proteins that interact with DnaK are enriched with high molecular weight proteins (> 60 kDa). Furthermore, the amount of protein that transiently interacts with GroEL was unaffected by changes in DnaK levels with or without TF. Therefore, the loading of GroEL with substrate does not require the presence of the DnaK system and TF. These findings indicate the existence of alternative folding pathways for newly synthesized proteins in E. coli. Some proteins are DnaK (probably ClpB)
While interacting with (when aggregated) other proteins interact with the GroEL system. Inducers as ribosome-associated chaperones also play essential roles in folding in both population types.

The role of chaperones in cell survival under heat stress conditions using the aforementioned ΔrpoH strain is of essential importance. This strain showed high heat sensitivity (Fig. 15).
. Expression of the DnaK system in ClpB was most effective in suppressing cell death at heat shock temperatures (Fig. 15), effectively preventing and retrogressing cellular protein aggregation (Fig. 15).
) As well as the heat labile reporter enzyme (firefly luciferase, FIG. 15). Increased production of the DnaK system and ClpB and its homologs is useful for increasing stress tolerance including survival and recovery from heat stress of wild-type as well as the ΔrpoH strain of E. coli. Therefore, DnaK
Overproduction of the system and ClpB homologs is also suitable for increased stress tolerance of other prokaryotic and eukaryotic cells.

Another result of the present invention is that Lon and ClpXP are the major cytoplasmic substrate proteases responsible for the elimination of aggregation prone proteins that accumulate in rpoH mutant cells. Therefore, it is useful for designing E. coli strains or E. coli-based in vitro protein synthesis systems for recombinant protein production. In particular, the elimination of these proteases increases the yield of labile aggregation-prone recombinant proteins.

The following examples are presented for purposes of illustration and are not intended, and should not be construed, as limiting the scope of the invention or the claims below.

Illustrative of the Invention Escherichia coli cytosolic chaperones may fold proteins into their native structure, prevent and reverse aggregation of heat-denatured proteins, and assist in refolding these substrates. Was investigated. Analysis of heat denaturation and aggregation of proteins is a well established experimental approach to study protein misfolding that also occurs during stress treatment of cells. With regard to the role of chaperones, temperature-induced aggregation of proteins resembles aggregation and inclusion body formation of recombinant proteins that frequently occur during bacterial overproduction. In addition, aggregation of heat-denatured proteins (eg, malate dehydrogenase, MDH) was found to increase staining with Congo-Red, a marker dye widely used to display amyloid fibrils. Congo-red in particular reacts with the anti-parallel β-sheet structure associated with fibrosis (Turnell and Finch, 1992). Therefore, aggregation of heat-denatured proteins increases the anti-parallel β-sheet component, and this change in secondary structure is shared by amyloid and prion-forming proteins. Therefore, analysis of heat-denatured proteins is considered relevant for studies of amyloid and prion metabolism.

For E. coli, the gene encoding the recombinant protein and ClpB and Dn
A set of compatiblities capable of independently controlling the expression of genes encoding the aK system.
e) The plasmid was constructed. Plasmids were also constructed encoding additional chaperone genes, particularly the GroEL-GroES system and inducers, which may also be useful for increasing the yield of soluble proteins in vivo.

In addition, an E. coli strain was constructed for high level production of labile recombinant proteins.
This strain harbors the plasmid described above and further has an IS element in the promoter region of the GroEL-GroES operon, which results in the lack of a major cytoplasmic substrate protease activity in cells capable of growth, a regulatory rpoH. Has a chromosomal deletion of the gene.

Example 1 DnaK Chaperone System is Effective in Preventing Protein Aggregation In this experiment, the DnaK chaperone system was assayed for its ability to prevent protein aggregation compared to other chaperone / chaperone systems. The DnaK system includes the heat shock protein DnaK (Hsp70), the cochaperone DnaJ (Hsp40), and the cochaperone GrpE. The GrpE cochaperone regulates the ATPase activity of DnaK and thus assists in the refolding of DnaK's protein substrate (Bu.
Kau and Horwich, 1998).

The experimental approach was based on published procedures (Hesterkamp and Bukau, EMBO J., 17
, 4818-4828), and relies on the identification of aggregated proteins by centrifugation. ³⁵ S
-A soluble extract of E. coli wild-type cells labeled with methionine (4 mg / ml),
Preincubation was performed at 30 ° C. for 5 minutes in the absence or presence of cytosolic chaperones. ATP (10 mM) was added 2 minutes before heat shock. The sample was then shifted to 45 ° C for 15 minutes. Soluble and insoluble protein material was separated by centrifugation. Aggregated proteins were pelleted by centrifugation at 15000g for 15 minutes at 4 ° C. Wash the pellet twice with ice-cold cutting buffer,
It was analyzed by one-dimensional and two-dimensional gel electrophoresis.

The amount of aggregated protein was measured by scintillation counting.
FIG. 1 compares the ability of different chaperones to prevent protein aggregation in E. coli cell extracts. As shown in FIG. 1, the DnaK system is an effective cytosolic chaperone system for the prevention of aggregation of heat-denatured E. coli proteins in cell extracts, and the chaperones and heat-labile proteins at approximately equimolar concentrations in cell extracts. Prevention of aggregation under substrate conditions Cytosolic chaperone GroEL / GroES, HtpG (Hs
p90), ClpB, and IbpB.

The results of this experiment showed the use of the DnaK system as an element in the technical approach to increasing the solubility of recombinant proteins.

Example 2: Identification of heat sensitive proteins whose aggregation is prevented by the DnaK system In vivo experiments were performed to identify more than 60 heat sensitive E. coli proteins whose temperature-induced aggregation is prevented by the DnaK system. The proteins thus identified are shown in Table 2. Δ after shifting to unacceptable heat shock temperature (42 ° C)
Proteins were identified by analysis of aggregated proteins that accumulated in dnaK mutants. E. coli wild type and dnaK mutant cells were grown exponentially in LB media at 30 ° C and heat shocked at 42 ° C for 60 minutes. Insoluble cell fractions (including membrane proteins) were prepared and analyzed by two-dimensional gel electrophoresis according to standard protocols. FIG. 2 shows aggregated proteins as Coomassie stained spots after two-dimensional gel electrophoresis of pellet fractions of wild type and dnaK ⁻ cells.

[0112]

[Table 2]

[0113] Figure 2 shows that protein aggregation at high temperature is greatly enhanced with the dnaK null mutant in vivo. This finding and reported Dna in wild-type cells
Due to the limited capacity of the K system (Tomoyasu et al., 1998), when the DnaK system is overproduced at the same time, the yield of soluble conformers of these proteins in bioengineering applications, eg after overproduction in E. coli, is increased. It is suggested to increase.

Example 3: Identification of the relative ability of various chaperones to disaggregate and refold aggregated proteins in vitro (combined action of the DnaK system and ClpB). The ability of various chaperones to disaggregate and refold aggregated proteins was identified. In vitro experiments were performed to achieve this. In a pair of experiments, total soluble extracts of E. coli wild-type cells were preincubated at 30 ° C for 5 minutes and heat shocked at 45 ° C for 15 minutes. Soluble and insoluble fractions were separated by centrifugation and analyzed by two-dimensional gel electrophoresis according to standard protocols. As shown in Figure 3, heat shock treatment increased aggregation of about 10-20% of the proteins in the lysate, which contained over 50 different protein species.

³⁵ S methionine labeled soluble extract of E. coli wild type cells was pre-incubated for 5 minutes at 30 ° C and heat shocked for 15 minutes at 45 ° C. Aggregated proteins were isolated by centrifugation and reaction buffer (50 mM HEPES (pH 7.6), 15
Resuspended in 0 mM KCl, 20 mM MgCl ₂ ). DnaK system (Dna
K, DnaJ, and GrpE), GroEL system (GroEL and G
roES), IbpB, HtpG, and ClpB (final concentration 2 μM).
), And ATP regeneration system were added under various conditions and incubated at 30 ° C. for 4 hours. Soluble and insoluble protein material was separated by centrifugation. The amount of soluble and insoluble fractions was determined by scintillation counting. As shown in FIG. 4, when added individually, these chaperones had no potential for singing aggregated proteins within 4 hours, even at high concentrations added to aggregated proteins. In contrast, when added with the ClpB and DnaK systems at a final concentration of 2 μM, about 50% of the aggregated E. coli protein was solubilized within 4 hours at 30 ° C. (FIG. 4).

A soluble extract of E. coli wild-type cells was preincubated for 5 minutes at 30 ° C.,
Heat shock was applied for 15 minutes at 45 ° C. The aggregated protein was isolated by centrifugation, and the reaction buffer (50 mM HEPES (pH 7.6) was added by pipetting.
, 150 mM KCl, 20 mM MgCl ₂ ). ClpB (2 μl) and DnaK (2 μM), DnaJ (0.4 μl) with ATP regeneration system
M), and a DnaK system consisting of GrpE (0.2 μM) was added. After incubation for 4 hours at 30 ° C., soluble and insoluble fractions were separated by centrifugation and analyzed by two-dimensional electrophoresis according to standard protocols. As shown in Figure 5, at least 70% of the different aggregated protein species showed increased solubility after incubation with the ClpB and DnaK systems (Figure 5).

Example 4 Heat Sensitive Proteins Disaggregate and Refold In Vitro with the DnaK System / ClpB Combination Heat Sensitive Test Proteins of Various Chaperones (Malate Dehydrogenase (
A pair of experiments were performed to analyze the ability to disaggregate and refold MDH) and firefly luciferase). Qualitatively similar results were obtained with all tested proteins, the results for MDH are summarized in Figure 6 and Table 3 and described in more detail below.

Incubation of MDH at 47 ° C. inactivated and formed large aggregates as judged by loss of enzyme activity, resulting in light scattering (turbidity) of protein solutions.
, And aggregates were detected microscopically. This is shown in FIG. 6A, which is 47
Mitochondrial M in the presence of DTT (10 mM) without chaperone at ℃
Time-dependent inactivation and aggregation of DH (720 nM) (increase in turbidity at 550 nm) is shown. As shown in Table 3 and Figure 6A, neither the ClpB with or without ATP and the DnaK system alone are active in MDH disaggregation and refolding. On the other hand, as shown in Table 3 and FIG. 6B (the above-mentioned heat treatments were performed with ClpB, DnaK, and concentrations of 500, 1000, 200, and 100 nM, respectively).
MDH agglomerated by heat treatment supplemented with DnaJ and GrpE at 25 ° C.
, Which shows time-dependent aggregation and reactivation at 3), is completely solubilized within 30 minutes by the combined ATP-dependent activity of the ClpB and DnaK systems, and almost completely reconstitutes up to 3 μM MDH within 3-4 hours. Activated

[0119]

[Table 3]

Description of Table 3: Aggregation rates were measured at either t = 0 '(t ₀ ) or t = 45' (t ₄₅ ). Unless indicated otherwise, the concentrations were as follows: MDH. agg, 0.7
2 μM; ClpB, 0.5 μM; DnaK, 1 μM; DnaJ, 0.2 μM; G
rpE, 0.1 μM; GroEL, 4 μM; GroES, 4 μM; hptG, 1
μM. ^* ClpB and ATP caused further aggregation rather than disaggregation. In the table, B means ClpB, KJE means DnaK system (DnaK, DnaJ, and G
rpE), LS is GroEL-GroES, and HscA / B is Hsc.
A and HscB (homologs of DnaK and DnaJ, respectively).

Examples 3 and 4 show that the ClpB and DnaK systems form a co-chaperone network that is highly effective in solubilizing and reactivating a wide range of aggregated proteins in vitro.

Example 5 In Vivo Activity of the DnaK System-ClpB Combination In vivo experiments were performed to determine if the combination of the DnaK system and ClpB was effective in reversing the formation of protein aggregates in E. coli. The protein disaggregation potential of wild-type cells was determined by clpB (Squires et al., 1991) or dna.
This was compared to the possibility of a mutant strain lacking any of K (Paek and Walker, 1987). E. coli wild-type, dnaK, and cplB mutant cells were grown exponentially at 30 ° C and heat shocked at 45 ° C to induce protein misfolding. Insoluble cell fractions (including membrane proteins) at the times indicated in Figure 7 (A) before and after heat shock were prepared by centrifugation. Thermolabile proteins were visualized and identified by Coomassie stained SDS-polyacrylamide gel electrophoresis (FIG. 7A) followed by immunoblotting (FIGS. 7B and C). In wild-type cells, the increased temperature resulted in only a small and transient accumulation of aggregated proteins. In ΔdnaK mutant cells, numerous and large numbers of different proteins irreversibly aggregated, as outlined in detail above. In the ΔclpB mutant cells, as shown in FIG. 7, a small amount of different proteins aggregated after the temperature was raised, but they did not aggregate within 3 hours. This aggregation behavior of cellular proteins for RNA polymerase and MetE protein subunits using immunodetection is described in more detail in FIG. 7C.

Based on these findings, to identify whether extensive aggregation of proteins in ΔdnaK mutant cells at 45 ° C. can be reversed by subsequent overproduction of the ClpB and / or DnaK system, In vivo experiments were performed. For this purpose, a plasmid was constructed so that the expression of each gene could be induced by the addition of IPTG to the culture medium. Therefore, it lacks the dnaK gene (ΔdnaK52
), DnaK, dnaJ, and clpB overexpressing plasmids (pdna
E. coli mutant cells with K / dnaJ / clpB) were grown exponentially at 30 ° C. The cells were heat shocked at 45 ° C. for 30 minutes and transferred again to 30 ° C. for 2 hours. Induction of chaperone synthesis was performed by the addition of IPTG (1 mM). IP
Further incubation in the absence of TG was used as a negative control. Insoluble cell fractions (including membrane proteins) were prepared and analyzed by two-dimensional gel electrophoresis according to standard protocols (Figure 8). As shown in FIG. 8, by inducing the expression of clpB and dnaK, dnaJ, and grpE, 70 to 80% of aggregated proteins were effectively solubilized within 2 hours at 30 ° C. These findings indicate that co-overexpression of the ClpB and DnaK systems can solubilize a wide range of aggregated proteins in vivo.

Example 6: Plasmid-based expression of ClpB, DnaK, DnaJ, and GrpE and GroEL-GroES chaperone system FIGS. 9A and 9B show independent recombinant proteins in E. coli containing ClpB, DnaK, DnaJ, and grpE. 1 shows a schematic of the plasmid-based expression capable of sexual co-overproduction as well as the GroEL-GroES chaperone system. Clp
B, a pair of common plasmids capable of independently regulating the expression of genes encoding the DnaK system, GroEL / GroES system, and recombinant target protein system were constructed.
This system is based on cassette-like plasmid elements and a modified arabinose-controlled expression vector (Mayer, 1995) developed by Bujard and coworkers (Lutz and Bujard, 1997).

The cassette system is used to express the chaperone gene so that the stoichiometry of each chaperone protein is adjusted to the normal stoichiometry found in wild-type E. coli cells. This adjustment results in optimal chaperone activity (Packschies et al., 1997) (unpublished observations). Depending on the cassette system, origins of replication (eg colEl, pl5A,
pSC101) is exchanged, thereby exchanging plasmid copy numbers of about 5 / cell to 50 / cell. Expression of the chaperone gene is driven by the IPTG inducible promoter Pall1a (Lanzer, 1998, Lutz et al. Bujard, 1997). Depending on the antibiotic resistance cassette, different resistance genes (eg Ap, Km, Cm, Sp
) Is selected. Arabinose-inducible PgAD with an arabinose regulatory vector
It is possible to express a gene of a recombinant target protein derived from a promoter (Mayer,
1995). Appropriate restriction sites allow the exchange of resistance genes and origins of replication for specific applications.

The expression system of the examples is only one possibility of simultaneous overproduction of chaperones and target proteins. Many different vector systems as well as chromosomally integrated expression systems are readily available to those of skill in the art.

Example 7: Protease-deficient recombinant molecule chaperone-based Escherichia coli producing strain useful for the production of high levels of recombinant protein In order to produce an unstable recombinant protein in E. coli at a high level, protease production is affected. A specific producer strain was designed that takes advantage of the published chromosomes that give This strain lacks a major cytosolic protease due to a deletion of the rpoH gene, which encodes the transcriptional activator δ ³² (Kusukawa and Yura,
1988). At the same time, it constitutively produces high levels of GroEL and GroES by transcriptional activation of the groES groEL operon by the insertion element. This strain was cloned into plasmid-based clpB, dnaK, dnaJ, grpE.
, GroES, and groEL as well as regulated overexpression of genes encoding recombinant proteins. This combination of protease deficiency and chaperone overproduction makes this strain very useful as a production strain in many biotechnology applications.

Example 8 Use of Chaperones in the Treatment of Diseases Associated with Protein Dysfunction Chaperones are useful in the prevention and reversal of protein aggregation associated with amyloidosis and prion diseases. Some neurodegenerative and age-related diseases, such as Kreuzfeld-Jakob disease and Alzheimer's disease, are due to the accumulation of misfolded proteins in the form of amyloid plaques, which are often cytotoxic. Similarly, environmental stresses such as heat shock can induce irreversible protein aggregation leading to cell death. According to the above example, E. coli ClpB and D
Molecular chaperone systems, including combinations with naK chaperones, have been shown to mediate active disaggregation and reactivation of aggregated proteins in vitro as well as in vivo. This novel combination provides the basis for research that can characterize the novel mechanism of intracellular defense of humans and other animals against protein post-folding and prion diseases, especially neuropathological amyloid diseases. Thus, the present invention provides the Hsp70 system and Cl in humans as a therapeutic design strategy for a number of diseases including, for example, prion-mediated diseases and age-related amyloid diseases.
It is intended to act as a pair of pB or Hsp100 homologs.

Example 9 Combination of Inducers Useful for Early Protein Folding with the DnaK System Neither the DnaK system nor the inducers are essential for the initial folding of a novel synthetic protein of E. coli (Hesterkamp and Bukau, 1998, unpublished). data). FIG. 10A shows the native δ ³² promoter, or tig ⁺ and Δtig :: kan.
FIG. 3 is a schematic diagram of the dnaK and dnaJ operons on the chromosome regulated by the background artificial IPTG inducible promoter PA 1 / lacO. Wild type, Δtig :: k
an, and DnaKiDnaJ regulatory Δtig :: kan and tig ⁺ cell cultures on LB plates with or without 1 mM IPTG at different dilutions (
10 ⁵ , 10 ⁴ , 10 ³ , 10 ² cfu / ml, 10 μl / spot). Cells were incubated at 30 ° C, 37 ° C, and 42 ° C for 24 hours and 9 ° C at 15 ° C.
Incubated for 6 hours. FIG. 10B shows that deletion of the inducer and DnaK is lethal to E. coli. DnaK / DnaJ deletion tig ⁺ and Δtig
Cultures of ::: kan cells were grown in M9 minimal medium in the mid-log phase (OD600 = 0.
Proliferated to 6) and pulsed with ³⁵ S-methionine (15 μCi / ml final concentration) for 15 seconds. Equal amounts of total protein (1.2 ml of 2 mg / ml total protein lysate) were centrifuged to isolate insoluble proteins and analyzed by two-dimensional gel electrophoresis according to standard protocols (FIGS. 11A and B). FIG. 11, which uses firefly luciferase (reporter protein) as the target protein,
In strains lacking the inducing factor (due to the deletion of the tig gene) and lacking DnaK (by IPTG-regulated blockade of expression of dnaK), the reporter protein (firefly luciferase) reduced folding efficiency, leading to large and large numbers of cells. It shows that the sex proteins cannot reach their natural state and cannot aggregate. Therefore, the combined action of both chaperone systems is very significant for the folding of newly synthesized proteins in E. coli. Furthermore, inducers and Dn in a wide variety of eubacteria
The presence of the aK homolog suggests that the collaboration between these two chaperones is generally important for protein folding in prokaryotic cytoplasmic substrates.

This knowledge is useful in improving the folding efficiency of prokaryotes by probable DnaK system of inducers and unnecessary overproduction. Furthermore, the folding efficiency of the protein product can be increased by adding an inducer to the in vitro protein synthesis system (in vitro translation system) currently developed for functional analysis of the genome.

Example 10: Identification of E. coli gene encoding ClpS-novel Hsp100 family cochaperone E. coli gene "yljA" which is the first gene in yljA-clpA operon.
Was cloned using standard protocols. yljA gene is 3
It is 18 bp long and encodes a 106 amino acid protein called "ClipS". FIG. 12 shows the DNA sequence of yljA (SEQ ID NO: 1) and the deduced amino acid sequence of the ClpS protein (SEQ ID NO: 2). These are identified in Table 4,
FIG. 13 shows the amino acid sequence alignment of ClpS homologs (SEQ ID NO: 3 to SEQ ID NO: 9). The proteins identified in Table 4 are BLAST and FASTA search results and representative putative ClpS homologs. It is interesting to note that the Helicobacter pylori (HP0032) homolog appears in the operon with the ClpA homolog, as in E. coli. Despite being 33% homologous to the whole protein, HP0032 is considered to be a ClpS homolog based on sequence identity and position.

Table 5 and Table 6 show the total sequence similarity of ClpS homologues, H-BOX, respectively.
Sequence similarity in regions (see Figure 13), regions conserved throughout evolution in ClpS homologs are shown. The sequences in FIG. 13 are the “Clustal” method (Higgin
s and Sharp, Gene, 15, 237-44, 1988) and sequence similarity in Tables 5 and 6 was determined by the "Megalign" program (Clewley and Arnold, Method).
S Mol. Biol. 70: 119-29, 1997).

[0133]

[Table 4]

[0134]

[Table 5]

[0135]

[Table 6]

Example 11: Establishing ClpS as a Cochaperone for ClpA Malate dehydrogenase (MDH) (0.9 μM) was aggregated by incubation for 30 minutes at 47 ° C in the absence of chaperone. Referring to FIG. 14, 0.5 μM ClpS after aggregation in the absence of chaperone (filled triangles).
(Black diamond), 0.5μM ClpA (open triangle), or 0.5μ
MDH activity in the presence of M ClpA + 0.5 μM ClpS (filled circles) was monitored. As shown in FIG. 14, MDH did not restore significant activity in the absence of chaperone or in the presence of ClpS alone. 3 in the presence of ClpA alone
Up to 30% MDH activity was obtained after 00 minutes. When ClpS was supplemented with ClpA, both the rate and the amount of MDH activity increased more than 2-fold. Therefore, C
lpS is established as a potent cochaperone for ClpA.

Example 12: Under DnaK / DnaJ-depleted conditions, protease-deficient cells accumulate aggregated proteins This experiment shows that proteases Lon and ClpXP have low chaperone content (
It is important to eliminate protein aggregates in E. coli cells with DnaK / DnaJ). Therefore, the yield of recombinant proteins produced in E. coli producing strains or in vitro translation systems can be improved by the use of variants lacking ClpXP and Lon.

A. BB7349 strain (PA1 / lacO-1 dnaK, dnaJ lacI
q), strain BB7353 (PA1 / lacO-1 dnaK, dnaJ lacI).
q hslVU), BB7357 (PA1 / lacO-1 dnaK, dnaJ)
lacIq clpPX-lon), BB7361 (PA1 / lacO-1)
Cells of dnaK, dnaJ lacIq ΔhslVU clpPX-lon) were grown overnight at 30 ° C. in LB medium containing 1 mM IPTG. The preculture was diluted 1000-fold with LB medium with or without 500 mM IPTG and further cultured until the late logarithmic phase (about OD600 = 1). Aggregated proteins were isolated from 8 ml of culture and membrane proteins were removed by washing with NP40. 3.2
Aggregated proteins from ml culture / OD600 were analyzed by SDS-PAGE and staining with Coomassie Brilliant Blue. Aliquots of whole cell lysate and aggregated protein fractions were quantified by Bradford assay. PA / lacO-1 K, J; BB7349, ΔVU; BB7353, ΔXP
lon; BB7357, ΔXP lon ΔVU; BB7361, +; 500
Culture in the presence of mM IPTG,-; Culture without IPTG.

B. A 1000-fold dilution of the culture was added to several concentrations of IPTG (0, 50, 50).
Further culture in LB containing 0 mM to mid-log phase (approximately OD = 0.5), 4
Further culturing was performed at 2 ° C for 2 hours. Aggregated proteins were isolated from 8 ml cultures and membrane proteins removed as described above. As above, 1.6 ml of culture / O
Aggregated proteins from D600 were analyzed by SDS-PAGE and quantified. The% total protein was calculated by dividing the amount of aggregated protein by the amount of total protein in the cells.

Example 13: Overproduction of the Clp3 and Dnak lines improves thermotolerance and luciferase refolding of rpoH and wild-type cells In this experiment (Figure 15), the ClpB and DnaK lines co-operated (i ) Cell viability at lethal heat shock temperature (rpoH mutant) and (ii) Heat labile reporter enzyme (firefly luciferase) in rpoH and wild type cells subjected to transient exposure to lethal heat shock temperature. Is shown to act to improve the reactivation of the.

A. (ΔrpoH cells); ClpB (ΔrpoH cells containing ClpB overproduced from the plasmid); KJE (DnaK, Dna overproduced from the plasmid)
J, GrpE containing ΔrpoH cells); KJE, ClpB (ΔrpoH cells containing DnaK, DnaJ, GrpE and ClpB overproduced from the plasmid) 250 mM inducing expression of the plasmids encoding ClpB, DnaK, DnaJ, GrpE. Grow at 30 ° C. in LB medium supplemented with IPTG.
Cells were treated at the indicated measuring points at 50 ° C. and plated on LB agar plates containing 250 mM IPTG. After 48 hours of incubation, colonies were counted and viability was identified. As a control, non-heat-treated cells were set to 100%.

B. WT (wild-type cell) containing arabinose-inducible luciferase; (Δrp
oH cells); ClpB (rpoH cells containing overproduced ClpB); KJE (
ΔrpoH cells containing overproduced DnaK, DnaJ, GrpE); KJE,
ClpB (ΔrpoH cells containing overproduced DnaK, DnaJ, GrpE, and ClpB); and ESL (ΔrpoH containing overproduced GroESL).
Cells) were grown in LB medium supplemented with 250 mM IPTG to induce luciferase for 30 minutes in the presence of arabinose (0.4%). Aliquots (identical OD, 100 ml) were incubated in the presence of tetracycline (100 mg / ml) and glucose (1%) for 0, 2.5, 5, 7.5 minutes at 50 ° C, followed by 10 minutes at 30 ° C. Further incubation. Recovery (%) was calculated by comparison with luciferase activity (100%) of non-heat treated cells.

The above description of the present invention has been presented for purposes of illustration and description. The preferred embodiments described herein above are intended to be available to those skilled in the art in various embodiments, with various modifications adapted to the particular application or use of the invention. It

Furthermore, the description is not intended to limit the invention to the form disclosed herein. One of ordinary skill in the art can recognize or ascertain many equivalents of the particular embodiments described herein using only routine experimentation. Such equivalents are intended to be within the scope of the following claims.

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407-436. Johnson, JL, and Craig, EA (1997). In Vivo Protein Folding: A Complex Pathway That Is Unconfused. Cell 90, 201-204. Kusukawa, N., and Yura, T. (1988). E. coli. Heat shock protein GroE: a key protective role against heat stress. Genes & Dev. 2, 874-882. Langer, T., Lu, C., Echols, H., Flanagan, J., Hayer, MK, And Hartl
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J, GrpE forms a cellular chaperone machine that can repair heat-induced protein damage. EMBOJ. 12, 4137-4144. Skowyra, D., Georgopoulos, C., and Zylicz, M. (1990). The dnaK gene product, the Hsp70 homolog, can reactivate heat-inactivated RNA polymerase in an ATP hydrolysis-dependent manner. Cell 62, 939-944. Sogo, Kazuyo et al. (1998). Chaperone expression plasmid, European Patent Application No. 981.
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Comparison with pB and HtpG. J. Bacteriology 180 (19): 5165-5172. Tomoyasu, T., Ogura, T., Tatsuta, T., and Bukau, B. (1998). DnaK and DnaJ levels are , Provides stringent regulation of heat shock gene expression and protein repair in E. coli. Mol. Microbiol. 30, 567-581. Turnell, WG, and Finch, JT (1992). The dye Congo red to the amyloid protein porcine insulin. Binding represents a novel homology between amyloidogenic peptide sequences. J. Mol. Biol. 227, 1205-1223. Veinger, L., Diamant, S., Buchner, J., and Goloubinoff, P. (1998). The small heat shock protein IbpB from Escherichia coli stabilizes stress-denatured proteins for subsequent refolding by the multichaperone network
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[Sequence list]

[Brief description of drawings]

FIG. 1 shows prevention of heat-induced protein aggregation by the cytosolic chaperone system in cell extracts.

FIG. 2 shows protein aggregation in wild type and DnaK mutant cells after heat shock at 42 ° C.

FIG. 3 is a diagram showing aggregation of thermolabile proteins in cell extracts.

FIG. 4 is a diagram showing the disaggregation of protein aggregation in vitro by a molecular chaperone system combining DnaK system and ClpB in comparison with other molecular chaperones.

FIG. 5: Gel electrophoresis showing disaggregation of protein aggregation in vitro by DnaK, DnaJ, GrpE, and ClpB.

FIG. 6 shows the heat inactivated malate dehydrogenases ClpB and D.
FIG. 6 shows naK-mediated disaggregation and reactivation.

FIG. 7 shows the disaggregation of aggregated proteins after heat shock in vivo in E. coli wild-type and dnak and clpB null mutant cells.

FIG. 8 shows the disaggregation of protein aggregates by DnaK, DnaJ, and ClpB in vivo.

FIG. 9 is a schematic diagram of a chaperone overproduction system in Escherichia coli.

FIG. 10 is a diagram showing the synthetic killing rate of DnaK / DnaJ-deficient Atig :: kan cells.

FIG. 11 is a diagram showing insolubility of existing and newly synthesized proteins.

FIG. 12 shows the nucleic acid sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of YljA protein.

FIG. 13 shows an amino acid sequence alignment of ClpS homologs.

FIG. 14 shows that the ClpA / ClpS system reactivates malate dehydrogenase.

FIG. 15 shows that overproduction of the ClpB and DnaK lines improves thermotolerance and luciferase refolding of rpoH and wild type cells.

[Procedure amendment]

[Submission date] December 4, 2001 (2001.12.4)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 38/00 A61P 25/02 4H045 48/00 25/14 A61P 25/02 25/28 25/14 35 / 00 25/28 43/00 105 35/00 C07K 14/00 43/00 105 14/245 C07K 14/00 C12N 1/15 14/245 1/19 C12N 1/15 1/21 1/19 15/00 ZNAA 1/21 5/00 A 5/10 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE) , LS, MW, MZ, SD, SL, SZ, TZ, UG ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Gorobinov, Pierre Israel Jerusalem 93855 Hadref Street 429 / 2 F term (reference) 4B024 AA01 BA08 BA80 CA02 DA 01 DA02 DA06 DA07 DA12 EA04 GA11 4B065 AA19X AA26X AA26Y AA41X AA46X AA50X AA53X AA77X AA80X AA88X AA90X AA91X AA93X AB01 AC14 BA02 CA24 CA28 CA31 CA43 CA44 4C084 AA07 AA13 BA44 CA04 DC50 MA02 NA14 ZA161 ZA221 ZB211 ZB261 ZC411 4C086 AA01 AA02 AA03 AA04 EA16 MA01 MA02 MA03 MA04 MA05 MA10 NA14 ZA16 ZA22 ZB21 ZB26 ZC41 4C087 AA01 AA02 BC34 BC83 CA12 MA02 NA14 ZA16 ZA22 ZB21 ZB26 ZC41 4H045 AA10 AA30 BA10 CA11 DA89 EA20 EA21 EA28 FA72 FA73 FA74

Claims (38)

[Claims] 1. A target protein comprising a first protein selected from the group consisting of at least one Hsp100 protein or a homolog thereof and a second protein selected from a group consisting of at least one Hsp70 protein or a homolog thereof. And a method of regulating the conformation of the target protein, which comprises regulating the conformation of the target protein. 2. The method of claim 1, wherein the first protein comprises a Clp homolog. 3. The method of claim 2, wherein the Clp homolog is ClpA, ClpB or ClpX. 4. The method of claim 1, wherein the second protein is DnaK containing a prokaryotic homolog. 5. The method of claim 1, wherein ClpB is used in combination with the DnaK chaperone system. 6. The method of claim 1, wherein the molecular chaperone system further comprises at least one cochaperone. 7. The method of claim 1, wherein the target protein is recombinantly expressed in a host cell. 8. The method of claim 1, wherein the first and second proteins are recombinantly expressed in a host cell. 9. The host cell is E. coli, S. The method according to claim 1, which is a cell of S. cerevisiae, Bacillus subtilis, plant, insect, yeast or mammal. 10. A GroEL encoding a first and a second protein and
-By transforming or transfecting a host cell with a vector capable of recombinantly expressing the GroES system or its homolog, the GroEL-GroES system protein or its homolog, or the inducer or its homolog, the first and second proteins are 9. The method of claim 8 which is recombinantly expressed. 11. The method of claim 1, wherein the target protein is aggregated or disaggregated in vitro. 12. A recombinant expression vector encoding at least one Hsp100 protein or homologue thereof and at least one Hsp70 protein or homologue thereof. 13. The vector according to claim 12, which is an inducible recombinant expression vector. 14. The vector of claim 12, further encoding at least one Hsp70 protein cochaperone. 15. The vector according to claim 12, which further encodes a protein of interest. 16. A cell transformed with the vector according to claim 12. 17. The cell according to claim 16, which is a protease-deficient prokaryotic cell or eukaryotic cell. 18. The cell according to claim 17, wherein the protease deficiency is caused by a mutation in the rpoH gene. 19. The cell according to claim 18, wherein the mutation is a chromosomal deletion of the rpoH gene. 20. The cell according to claim 16, wherein the cell recombinantly expresses a protein of interest. 21. The cell according to claim 20, wherein the protein of interest is malate dehydrogenase. 22. Aggregation, comprising the step of contacting the aggregated protein with a molecular chaperone system containing at least one Hsp100 protein or homologue thereof and at least one Hsp70 protein or homologue thereof, thereby disaggregating the aggregated protein. In vitro disaggregation method of the obtained protein. 23. A set comprising co-expressing a molecular chaperone system containing at least one Hsp100 protein or homologue thereof and at least one Hsp70 protein or homologue thereof, thereby increasing the solubility of the recombinantly expressed protein. A method for increasing the solubility of a recombinantly expressed protein. 24. At least one Hsp100 protein or homologue thereof and at least one for preventing protein misfolding and aggregation.
A method of preventing cell death caused by misfolding and aggregation of proteins, comprising the step of administering a therapeutically effective amount of a molecular chaperone system containing one Hsp70 protein or a homologue thereof, thereby preventing cell death. 25. The method of administering a therapeutically effective amount of a molecular chaperone system containing at least one Hsp100 protein or homologue thereof and at least one Hsp70 protein or homologue thereof, thereby treating a disease A method of treating animal diseases caused by folding and aggregation. 26. The disease is Creutzfeldt-Jakob disease, Alzheimer's disease, Huntington's disease, ataxia type 1, cystic fibrosis or cancer.
The method according to 5. 27. A pharmaceutical composition comprising an effective dose of a molecular chaperone system comprising at least one Hsp100 protein or homologue thereof and at least one Hsp70 protein or homologue thereof, and a pharmaceutically acceptable carrier. 28. A method of increasing the folding efficiency of a target protein in vitro, comprising the step of adding an inducer to the in vitro protein synthesis system, thereby increasing the folding efficiency of the protein in vitro. 29. The method of claim 28, further comprising the step of adding to the in vitro synthesis system a molecular chaperone system containing at least one Hsp70 protein or homologue thereof. 30. A method of increasing protein folding efficiency in a cell, comprising the step of recombinantly expressing an inducer in the cell, thereby increasing the protein folding efficiency in the cell. 31. The method further comprising the step of recombinantly expressing a molecular chaperone system containing at least one Hsp70 protein or a homologue thereof.
The method described in 0. 32. (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1; (b) a polynucleotide which is antisense to the polynucleotide of (a); (c) (a) or (b) A polynucleotide comprising 80% identity to the polynucleotide; (d) a polynucleotide encoding the protein of SEQ ID NO: 2; (e) a 80% identity to the protein of SEQ ID NO: 2. A polynucleotide encoding a polypeptide having identity; (f) to a polypeptide (a) to (e), which encodes a protein capable of modulating the activity of Hsp100 protein or a homologue thereof in disaggregation 80
% Polynucleotide, and (g) a polynucleotide which hybridizes under stringent conditions to any one of the polynucleotides described in (a) to (f), An isolated polynucleic acid molecule selected from: 33. (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; (b) a polypeptide having 80% identity to the amino acid sequence of SEQ ID NO: 2; (c ) A polypeptide variant of the amino acid sequence of SEQ ID NO: 2; (d) A polypeptide variant of the amino acid sequence of SEQ ID NO: 2 which can regulate the activity of the Hsp100 protein or its homolog in disaggregation; (e) Assist ClpA A polypeptide variant of the amino acid sequence of SEQ ID NO: 2; (f) a polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO: 1; (g) 80% of the sequence of SEQ ID NO: 1 A polypeptide encoded by a polynucleotide having identity; and (h) a polynucleotide having the sequence of SEQ ID NO: 1. An isolated polypeptide selected from the group consisting of: a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with respect to. 34. The polypeptide of claim 33, wherein the variant is an insertion variant, a deletion variant, or a substitution variant. 35. A first protein selected from the group consisting of at least one Hsp100 protein or homologues thereof, and at least one Hs.
To treat a disease, which comprises a step of administering a pharmaceutical composition comprising a gene therapy vector encoding a molecular chaperone system comprising a second protein selected from the group consisting of p70 protein or homologues thereof Gene therapy. 36. A first protein selected from the group consisting of at least one Hsp100 protein or homologues thereof, and at least one Hs.
36. The composition for gene therapy according to claim 35, comprising a gene therapy vector encoding a second protein selected from the group consisting of p70 protein or homologues thereof. 37. A first protein selected from the group consisting of at least one Hsp100 protein or homologues thereof, and at least one Hs.
An isolated molecular chaperone system comprising a second protein selected from the group consisting of p70 protein or homologues thereof. 38. A method for regulating the activity of Hsp100 protein or a homologue thereof, comprising the step of contacting Hsp100 protein with the polypeptide according to claim 33.